Parathyroid hormone (PTH), acting via PTH/PTHrP receptors (PTHRs), regulates bone cell activity in a complex manner. Intermittent (once daily) administration increases bone mass, whereas continuous treatment causes net bone resorption. PTHRs simultaneously activate several different intracellular effector pathways, including adenylyl cyclase (AC), phospholipase C (PLC) and protein kinase C (PKC). This project addresses the hypothesis that the anabolic and catabolic effects on bone of intermittent vs. continuous PTH reflect different patterns of activation of these effector pathways. We have developed unique analogs of PTH that preferentially activate or suppress generation of AC-independent signals via the PTHR, as well as mutant (DSEL) PTHRs selectively defective in PLC/PKC signaling. These tools enable us to dissect contributions of these pathways, relative to that of AC/cAMP signaling, to PTH regulation of bone formation and resorption. At doses that comparably activate AC, these "signal-selective" analogs will be administered once daily (4 weeks) or continuously (2 weeks) to intact male or ovariectomized female mice. Skeletal responses at different sites will be assessed by DEXA, microCT and dynamic histomorphometry, and changes in serum biochemistry, bone markers and skeletal gene expression will be measured. Alterations in numbers of marrow osteoprogenitors induced in vivo will be sought using ex vivo marrow cultures, and mechanisms of observed differences further analyzed by treatment of normal marrow cells with PTH or analogs in vitro. Signal-specific differences in regulation of osteoblast proliferation, differentiation and apoptosis will be assessed using established cell lines, including PTHR-null cells reconstituted with either wild type or DSEL mutant PTHRs, to study contributions of PLC/PKC signals in defined in vitro systems. Roles of ERK1/2, Ras/Raf and Rap-1/B-Raf signaling in proliferative responses, and of p38 MAPK in osteoblastic differentiation, will be specifically addressed, and the possibility that different PKC isoforms may be activated by PLC-dependent vs. PLC-independent signaling will be examined. Preliminary evidence in DSEL knock-in mice that PLC/PKC signaling is important for PTH regulation of osteoclastogenesis will be further addressed using different PTH analogs in the continuous-treatment protocols in vivo and in cultures of intact calvarial bone, normal femoral marrow or wild-type vs. DSEL PTHR-expressing marrow stromal cells in vitro. These studies will provide important new information concerning the manner and extent to which specific PTHR-generated messenger signals regulate cellular activity in bone and may thereby open new avenues to the design of PTH analogs with improved net anabolic activity.